The stone crayfish, Austropotamobius torrentium, is a native European freshwater species sensitive to habitat alterations such as stream channelization and urban development, putting its populations at risk. This study aims to assess how habitat alteration and subsequent water quality changes affect the stone crayfish population in the Kustošak Stream (Croatia) through three selected sites under different levels of anthropogenic disturbance: (1) undisturbed; (2) recently modified, and (3) long-time modified sites. At each site, crayfish were captured, measured, and marked to estimate population size, structure, and crayfish condition. Additionally, we examined whether water quality (18 measured physicochemical parameters) affects relative crayfish abundance. We also used mitochondrial sequencing and microsatellite genotyping to assess species genetic diversity and population connectivity and to compare results among the sites. The results showed that habitat alteration caused an overall reduction in population abundance and changes in size structure; however, it had not yet resulted in detectable genetic differences. Partial least squares regression showed that crayfish abundance was affected by the physicochemical parameters of water, including, among others, oxygen, calcium ions, nitrates, pH, and water temperature. We discuss our findings in the context of the effects of anthropogenic disturbance on the viability and persistence of this EU priority species.