The main parameter to verify the quality of milk is the microbiological profile and microbial contamination index. The aim of this study was to isolate and identify gram-negative proteolytic bacteria in different dairy production systems in western Paraná, Brazil. Thirty-five milk samples collected directly from milk tanks evaluated for flow cytometry by somatic cells count (SCC) and total bacteria count (TBC). Samples were diluted in peptone water and seeded in caseinate agar. The colonies were inoculated into Escherichia coli broth for 48 h at 35°C. Subsequently were inoculated Eosin Methylene Blue Agar (EMB) and inoculated in Bactray I and II Kit, for the purpose of identification of gram-negative oxidase negative bacteria. Twenty 7 samples showed isolation and identification of some kind of agent, with 77.1% of bacteria isolated. The mean values obtained for TBC and SCC were 1.867.000 cfu/mL and 944.000 cels/mL respectively, values considered above the current legislation. The frequencies of isolated species were Escherichia coli (8.6%), Escherichia fergusoni (8.6%), Klebsiella oxytoca (8.6%), Yersinia enterocolitica (8.6%), Hafnia alvei (5.7%), Serratia liquefaciens (5.7%) Serratia odorifera (5.7%), Citrobacter freundi (2.85%), Klebsiella ornithinolytica (2.85%), Klebsiella pneumoniae (2.85%), Proteus mirabilis (2.85%), Providencia rustigiani (2.85%), Providencia Stuart II (2.8 5%), Salmonella (2.85%), Serratia rubidaea (2.85%) and Shigella flexneri serogroup B (2.85%). Of isolates identified 42.85% of them belonged to the family Enterobacteriaceae, poor hygiene indicators in manufacturing processes. Proteinases produced by this microbiota are thermostable, remains intact and active after heat treatment, and the considerable economic losses, and one should trace their sources of contamination as hazards to public health.