The integrity of rivers is affected by anthropogenic activities at different spatial scales, from basin and landscape levels to the direct effects on the river and aquatic life. Our objective was to study these effects on the subtropical La Pasión River, analyzing environmental, geomorphological, habitat and water quality, and macroinvertebrates. We sampled the dry season (March 2022) because the river presented stable conditions. We selected the most influential variables in each spatial scale and determined their relationship with the indexes of quality characteristics and aquatic life in the river using multivariate statistics. Most sites (≈65%) had medium water and suboptimal habitat quality status, meanwhile half the sites had regular biotic integrity status; without finding coincidence in the quality of the different indexes applied, all sites indicated a high gradient of degradation from the origin to the mouth of the river. The presence of some families (e.g., Culicidae, Chironomidae, Lumbriculidae) indicated organic matter contamination. The main variables that significantly classified the river quality and integrity structure were water flow, turbidity, habitat embeddedness, and sulfates (χ2 = 0.1145, p < 0.01). It is concluded that the affected sites received wastewater without prior treatment and presented physical barriers such as irrigation channels.