Introduction. There is evidence of a connection between infectious and inflammatory lesions of the lower urinary tract and bladder cancer (BCa). However, there is virtually no data on the urine microbiota of middle-aged and elderly men with suspected BCa. This knowledge is extremely important from the point of view of studying the role of the infectious-inflammatory hypothesis in the genesis of BCa.Objective. To conduct a comparative assessment of the microbiota of bladder urine obtained during natural urination and bladder catheterization through standard and extended cultural studies in men with a presumptive diagnosis of non-muscle-invasive bladder cancer (NMIBC).Materials & methods. The prospective comparative study with consecutive patient recruitment included 23 men older than 45 years with suspected NMIBC based on clinical, laboratory and sonographic data, as well as with no history of infectious and inflammatory diseases of the kidneys and urinary tract, including sexually transmitted infections and recurrent infections of other organs and systems. A midstream urine samples and catheter-drained urine immediately before urethrocystoscopy were subjected to bacteriological analysis. Urine culture study was carried out using a standard set of nutrient media under aerobic cultivation conditions and an expanded set of nutrient media under aerobic and anaerobic cultivation conditions.Results. An anaerobic spectrum of microorganisms was predominantly detected in the urine of patients with suspected NMIBC. Moreover, Peptococcus spp. was more often found among anaerobic taxa. (70%), Eubacterium spp., Propionibacterium spp. (45% each, respectively), among representatives of the aerobes — Corynebacterium spp. (60%), S. lentus (up to 45%), S. haemolyticus (35%) and E. faecalis (30%). Data from a comparative analysis of the detection frequencies of microorganisms depending on the method of collecting material and the set of media for cultivation showed that more isolates are isolated in the midstream urine samples than in the study of catheter urine (2.9- and 1.9-fold with the standard and extended sets of media, respectively). In addition, when performing an extended bacteriological study compared to the standard method, the detection of microorganisms is 3.5-fold higher when examining an average portion of urine and 5.1-fold higher when examining catheter urine. The average number of microorganisms per one patient is also higher when using the extended method (midstream portion — 4.7 ± 1.9; catheterised urine — 2.3 ± 1.0) in comparison with the standard one (midstream portion — 1.3 ± 0.9; catheterised urine — 0.3 ± 0.5).Conclusion. Middle-aged and older men with NMIBC are characterized by asymptomatic bacteriuria in the form of a wide range of aerobic and anaerobic microorganisms. The urine pattern obtained with a urethral catheter contains a significantly narrower range of bacteria compared to the sample obtained through natural urination. It is advisable to carry out an extended cultural urine examination to study the characteristics of the microbiota / microbiome of the urine of the bladder immediately before the urethrocystoscopy procedure by collecting urine with a urethral catheter.