Freshwater systems are among the most threatened ecosystems worldwide, facing degradation from multiple anthropogenic pressures, including the introduction of emerging contaminants such as pharmaceuticals and personal care products (PPCPs). To evaluate the occurrence, distribution, and ecological risk of PPCPs across environmental compartments, a multi-matrix study was conducted along the Elsa River (Tuscany, Italy). Seventeen PPCPs, spanning six therapeutic classes, were quantified in surface water, bed sediments, and muscle tissue of the bioindicator fish (Squalius squalus) across seven sampling stations influenced by agricultural, industrial, and urban inputs, including three municipal wastewater treatment plants (WWTPs). Concurrent microbiological analyses assessed effects on microbial community structure and abundance. Ten PPCPs were detected across the environmental matrices, with steroid hormones and analgesics/antipyretics being the most prevalent. Contamination levels increased from upstream to downstream, particularly in water and sediments, after WWTP discharge points. Caffeine was found at all sites and across all matrices, serving as a clear indicator of continuous anthropogenic pressure along the entire river. The steroid hormones almost consistently exceeded the proposed WFD EQS limits in nearly all matrices and sampling sites. Risk quotient (RQ) analysis showed low risk (RQ < 1) for most compounds in water, though ibuprofen and steroid hormones frequently reached RQ >1. In sediments, amoxicillin, ibuprofen, caffeine, and steroid hormones posed high risks to benthic organisms, while all steroid hormones exhibited high RQs in biota. Microbiological assays revealed some shifts in microbial communities concurrent with elevated PPCP loads. These findings confirm WWTPs as principal contaminant sources and underline the urgent need for integrated multi-compartment monitoring, wastewater treatment upgrades, and targeted mitigation.