Despite good manufacturing practices and rigorous cleaning and sanitizing procedures established in dairy processing plants, microbiological contamination remains the main cause of products being non-compliant and/or atypical and hence not fit for human consumption. The objective of this study was to isolate, identify and characterize bacteria, yeasts and molds associated with substandard dairy products in Canada and to create a collection of reference isolates. In addition to conventional microbiological characterization, each isolate was tested for biofilm-forming ability and susceptibility to heat, antimicrobial agents, and common industrial disinfectants. Among the 105 microbial strains isolated from pasteurized milk, cream, and cheese samples, 24 bacterial isolates, belonging mainly to the genus Pseudomonas, were shown to be moderate or strong biofilm producers in 96-well plates and highly resistant to peracetic acid (100 ppm, 5 min contact time) and sodium hypochlorite (70 ppm, 5 min contact time). In addition, 56 bacterial isolates, including Acinetobacter baumannii, Enterobacter bugandensis, Klebsiella pneumoniae and Pseudomonas spp., were found resistant to ampicillin, fosfomycin and/or ceftriaxone, while 14 others, such as Bacillus spp. and Macrococcus spp., withstood a heat treatment equivalent to low-temperature long-time pasteurization (63°C for 30 min). This descriptive study provides new information on potential problematic microorganisms in dairies and will guide the development of novel control strategies intended to prevent and reduce microbiological contamination and the associated economic losses.