River contamination by microorganisms, or another chemical source, poses a serious threat to both the environment and public health. Taking immediate and appropriate actions is essential to mitigate the contamination and prevent further spread. As such, regular monitoring of these pollution agents is essential to act in time and control its minor extension. However, there is a lack of commitment to this emergent concern and respective actions around the world. This work aims to study the contamination of a Portuguese river (Tinto River) within Porto city (a highly populated urban area) regarding the total aerobic microorganisms, coliforms, and Enterococcus (as colony-forming units (CFUs) using specific solid culture media) and total organic matter (TOC). Different locations were considered along the Tinto River course (i.e., 14 locations within 11 km) and samples were collected on distinct days throughout September 2022. The overall results showed microbial contamination of aerobic microorganisms (up to 2 × 105 CFU/100 mL), total coliforms (up to 7 × 104 CFU/100 mL), Escherichia coli (up to 9 × 103 CFU/100 mL), and Enterococcus (up to 8 × 103 CFU/100 mL). The results also surpassed the maximum recommended values (MRVs) described in Portuguese decree-law no. 236/98 for irrigation waters. Moreover, TOC was found in a range of 4.54 mg/L to 57.2 mg/L. This work highlights the dangerous microbial contamination and higher amount of organic matter than would be expected for a surface water resource.