The quality of raw milk is important in determining the quality of manufactured dairy products. In recent years, the amalgamation of small dairy manufacturing plants into large mega sites has resulted in longer storage of milk during transportation and storage, particularly during peak milk flow in a seasonal dairy industry. The aim of this research is to determine the influence of this extended storage on psychrotrophic enzyme-producing bacteria even if the cold chain is maintained. In this study, the numbers and types of psychrotrophs in raw milk with and without refrigerated enrichment (5 days at 7 °C) were compared to understand the effect of extended refrigeration on the changes in raw milk microbial ecology. Two-hundred and six isolates from both fresh and enriched raw milk were identified by MALDI-TOF MS and assessed for proteolytic and lypolytic activity at 7 °C and the heat stability of these enzymes (72 °C, 15 s) was measured. This is the first report to compare the raw milk microbiota before and after extended refrigeration at both genus and species levels. This study also characterized these bacterial isolates, some of which have never been reported in milk, for their potential to spoil dairy products. •Extended refrigeration can shift the microbial diversity of raw milk.•Five Pseudomonas species producing heat stable protease and lipase found for the first time in milk.•MALDI-TOF MS and agar diffusion assays rapidly identifies protease and lipase producing bacteria.