In Lake Biwa, the largest lake in Japan, external pollutant loads have decreased since the 1980s, leading to improved water quality, such as reduction in biochemical oxygen demand (BOD) and PO43− concentrations. We examined long-term variation of bacterial production (BP) under these environmental changes from 2016–2017 in the north basin of Lake Biwa. BP was estimated by measuring the incorporation of stable isotope 15N-labeled deoxyadenosine (15N-dA) from June 2016 and December 2017 and compared with measurements from 1986 and 1997–1998. In 1986, BP was measured by following 3H-labeled thymidine (3H-TdR) incorporation and in 1997–1998 by tracking bacterial abundance in incubations and calculating specific growth rates (μ). To allow direct comparison of 15N-dA and 3H-TdR incorporation rates, we determined a conversion factor. To estimate μ in 2016–2017, we determined a factor for converting 15N-dA incorporation to cell number increase. In 2016–2017, the 15N-dA incorporation rate ranged from 0.13 to 30.7 pmol l−1 h−1 and μ ranged from 0.016 to 0.70 day−1. BP values from 3H-TdR incorporation rates in 1986 and μ in 1997–1998 were 4.6 and 2.1 times BP values in 2016–2017, respectively, confirming the decrease in BP over the past 3 decades in Lake Biwa. Water quality data showed only low decrease rates for BOD and total phosphorus concentration from the 1980s, whereas the rate of decrease for PO43− concentrations was equivalent to that of BP. BP and decomposition of organic matter are known to be strongly P-limited in Lake Biwa. Our results suggest that the decrease in BP can be explained by a reduction in readily bioavailable PO43−. Organic phosphorus can also be an important P-source for BP under conditions with very low PO43− concentrations (nM), and changes in the bioavailability of organic phosphorus might have also regulated BP dynamics.