Microbes play a central role in the decomposition and remineralization of organic matter and recycling of nutrients in aquatic environments. In this study, we examined the influence of physical, chemical, and biological parameters on the rate of bacterial production (BP) and viral production (VP) with respect to primary production over a diurnal period in Cochin estuary. Time series measurements were made every 2 h for 12 h (6 a.m.-6 p.m.) during periods of low and high salinities. The light intensity as photosynthetically active radiation, temperature, salinity, nutrients like NO3-N, SiO4-Si, and PO4-P, and chlorophyll a (Chl a) were measured along with BP, VP, and net primary production (NPP). NPP showed a strong positive correlation with light and Chl a (r (2) = 0.56 and 0.47, respectively), while VP showed a strong positive correlation with light, salinity, and Chl a (r (2) = 0.37, 0.58, and 0.37, respectively) and a negative correlation with BP (r (2) = -0.39) at P ≤ 0.05. We observed a diurnal pattern in BP but did not have any significant correlation with light. Similar diurnal pattern was seen in VP, the peak of which was in succession with BP, suggesting that virus-mediated lysis plays an important role in loss processes of bacteria in Cochin estuary. The results of our study highlight the light-dependent and physicochemical-dependent diurnal variation in virioplankton production in a tropical estuarine ecosystem.