Rivers bring large amounts of water, sediment, and other abundant materials (nitrogen, phosphorus, carbons, etc.) into lakes, which places great pressure on lake ecosystems. In this study, a novel bio-assessment framework integrating the bacteria-based index of biotic integrity (Ba-IBI) and path model was established to determine the direct and indirect effects of fluvial inputs on the ecological status of Dongting Lake, China. Twenty-two sampling sites were selected in Dongting Lake basin during two days in August 2017. After range, responsive, and redundancy tests, four core metrics were selected from the dataset of 130 candidate metrics to develop the Bairn, including the proportions of Flavobacteriia, Burkholderiales, phototrophs, and streptomycin producers. The Ba-IBI value, which was the sum of each individual metric value, was ranged from 1.37 to 3.66. Based on the 25th and 75th percentiles of the Ba-IBI, three levels were classified to show the influence degree of fluvial inputs on the ecological status. The Ba-IBI showed a gradient distribution for the distance from estuaries to the major lake regions. To further investigate the relative contributions of each individual fluvial input to the Ba-IBI, a path model was established and validated. The results showed that water nutrients (r = - 0.658, p < 0.1) was the controlling group of factors for the Ba-IBI, including direct (r = - 0.538, p = 0.002) and indirect effect (r = -0.12, p < 0.1). Water organics and sediment characteristics groups exerted direct effects, with coefficients of - 0.324 (p = 0.015) and 0.271 (p = 0.089). The hydrological parameters group (r = - 0.3, p < 0.1) had an indirect effect on the Ba-IBI mediated through water nutrients, water physicochemical properties, and sediment characteristics groups. The bio-assessment framework can provide a new insight for the management and protection of lake ecosystems.