Microbiological biopreparations have been successfully used for years in wastewater treatment plants for various purposes, including the removal of biogenic compounds such as nitrogen and phosphorus, acceleration of the biodegradation of organic substances, improvement of anaerobic processes, elimination of unpleasant odors, enhancement of activated sludge quality, and reduction of sludge volumes requiring disposal. Some biopreparation manufacturers also claim that these products can be effectively used for the revitalization of treated wastewater and for the “effective removal of pathogenic bacteria, pathogens, and parasites from wastewater and sewage sludge.” This study evaluated the effectiveness of the biopreparation ACS ODO_1 in eliminating coliform bacteria from treated wastewater, analyzing the impact of different preparation dosages and oxygen conditions. Both traditional culturing methods (fermentation-tube, membrane filtration, and pour plate techniques) and modern techniques (ATP luminometry, flow cytometry [FCM], and molecular PCR analysis) were used for assessment. The results of classical culturing methods showed that the greatest reduction of coliform bacteria was achieved in samples with the addition of the preparation combined with aeration, and the effectiveness increased with higher biopreparation doses. However, not all conditions led to a reduction in indicator microorganisms; in some cases, an increase in the number of coliform bacteria was observed, possibly indicating selective proliferation of undesirable microflora or inappropriate environmental conditions. Compared to traditional culturing methods, modern detection techniques provided different insights. ATP values and the number of viable cells detected by FCM were high in all samples, regardless of culturing method results. This may indicate the presence of microorganisms undetectable by traditional techniques (e.g., bacteria in the VBNC state) or the presence of active microflora introduced with the biopreparation. The interpretive differences stem from the fact that classical methods identify only bacteria capable of growing on selective media, whereas ATP and FCM reflect the total biological activity of the sample. The results suggest that a comprehensive evaluation of hygienization effectiveness and actual microbiological risk requires a combined application of both classical and modern methods. Only their complementarity allows for a reliable analysis of microbiological threats and an accurate assessment of the real activity of microorganisms present in wastewater.