Fish is an indispensable source of protein, omega-3 fatty acids, and critical nutrients. However, its quick perishable characteristics demand a proper storage strategy to extend shelf life. Refrigeration is one of the earliest methods of storage where temperature is maintained between 2 °C and 5 °C. Temperatures below 5 °C significantly regulate microbial growth and spoilage in most food products, but psychrotrophs thrive at these temperatures. Thus, it is essential to thoroughly identify and understand the development patterns of specific spoilage bacteria (SSB). The study aims to evaluate the impact of SSB activity on rohu quality assessed under refrigeration over 11 days. The study observations identified Aeromonas hydrophila as SSB. Also, observed bacterial load was found to correlate directly with spoilage parameters such as TMA, TVBN, PV, FFA, and TBARS, confirming the relationship between bacterial load and the progression of spoilage. Next, the role of SSB in fish spoilage was validated through controlled SSB inoculation experiments. Here, refrigerated rohu spoiled faster than the control groups, indicating the bacteria responsible for the deterioration of refrigerated rohu. Furthermore, the predominance of A. hydrophila over other microbes confirms its role as the primary SSB. Thus, monitoring and controlling SSB is crucial for understanding its growth dynamics, which assists in developing targeted strategies and effective control measures to preserve the organoleptic qualities of refrigerated products.