Microplastics are one class of widely prevalent emerging contaminants that have a detrimental effect on ecosystems and human health. The status of microplastic pollution in rivers in Western India is not well documented, making it difficult for establishing monitoring policies and guidelines. Mula river in Pune is a lifeline for the numerous industries and residential establishments in the city and hence monitoring the health of the water of Mula river is of great societal relevance. This investigation elucidated the prevalence, abundance and characterization of microplastics in Mula River water, while it crosses Pune, one of the fastest developing cities in India. Season played a determinant role in microplastic abundance. During the pre-monsoon season, the average microplastic concentration at all the three selected locations was notably higher (1808 ± 697 particles/L) compared to the post-monsoon period (1561 ± 167 particles/L). Microplastics smaller than 100 μm were consistently dominant in both the seasons. Notably, the most common polymer found in the Mula River was Ethylene Vinyl Acetate (EVA), a copolymer of polyethylene (PE) and vinyl acetate, commonly used in plastic wraps and packaging material. Unregulated disposal of industrial waste emerged as the most potential source of microplastics in Mula river. This study addresses a critical knowledge gap about the distribution and sources of microplastics in rivers in India. This work provides baseline data that can be used to access accurate mitigation of microplastics and evaluate health and environmental impacts of microplastic pollution in Indian rivers.