Microbial multi-enzyme production is a promising research area involving microbes that can simultaneously synthesize numerous enzymes for the degradation of substrates with environmental sustainability and other industrial applications as the focus. During this study, we isolated, screened, and identified some multi-enzyme-producing microbes from three soil sites in Ibadan: Apete Dumpsite (ADS); Botanical Garden University of Ibadan- (BGUI); and Eleyele Cassava Processing Field- (ECPF). Precisely 500 g of soil were collected per point at different depths (0–10, 10–20, and 20–30 cm) and further analyzed. The microbial load and type and enzyme assays were determined. The highest average microbial count (6.9 × 108 CFU/g) was recorded in the ECPF soil sample at 0–10 cm depth, while the lowest microbial count of 3.3 × 105 CFU/g at 20–30 cm soil depth in BGUI. Multi-enzyme-producing microbes were identified phenotypically and molecularly. This study revealed an inverse relationship between microbial community composition and soil depth. Bacterial and fungal genera had higher species richness, biodiversity, and evenness than yeast from all sampled points. With significant amylolytic, proteolytic, lipolytic, cellulolytic, and pectinolytic activities, 22 of the 193 isolates were identified as multienzyme producers. The blasted 16S rRNA and ITS rRNA amplicon sequences compared with sequences in the NCBI gene bank identified Bacillus, Roseomonas, Aeromonas, Phanerochaete, Aspergillus, Fusarium, Trametes, and Rigidoporus species. Results from this study show that topsoils (0–10 cm), rich with organic matter, are good reservoirs of multienzyme-producing microorganisms- applicable for environmental sustainability and in other important industries for waste management, food processing, and biomaterial production.