The activated sludge (AS) process is the most common type of secondary wastewater treatment, applied worldwide. Due to the complexity of microbial communities, imbalances between the different types of bacteria may occur and disturb the process, with pronounced economical and environmental consequences. Microscopic inspection of the morphology of flocs and microorganisms provides key information on AS properties and function. This is a time-consuming, highly skilled, and expensive process that is not readily available in all locations. Thus, most wastewater-treatment plants do not carry out this essential analysis, resulting in frequent operational faults. In this study, we develop a novel deep learning (DL) object detection algorithm to analyze and monitor the AS process based on a unique microscopic image database of flocs and microorganisms. Specifically, we applied YOLOv5 and Faster R-CNN algorithms as tools for segmentation and object detection to analyze the wastewater. The mean average precision (mAP) of the YOLOv5 was 0.67, outperforming the Faster R-CNN by 15%. Histogram equalization preprocessing of both bright-field and phase-contrast images significantly improved the results of the algorithm in all classes. In the case of YOLOv5, the mAP increased by 16.67%, to 0.77, where the AP of protozoa, filaments, and open floc classes outperformed the previous model by over 20%. These results demonstrate the potential of leveraging DL algorithms to enhance the analysis and monitoring of WWTPs in an affordable manner, consequently reducing environmental pollution caused by contaminated effluent. The fundamental challenge addressed herein has important global relevance, especially in an era in which the demand for high-quality wastewater reuse is expected to increase dramatically.