Nanoplastics, commonly used in cosmetics, enter wastewater systems and interact with activated sludge, yet their effects on microorganisms, essential for wastewater treatment, remain poorly understood. The aim of this study was to investigate the effects of polyacrylic acid-based nanoplastics (PANPs) on microorganisms in activated sludge. The PANPs were characterized in terms of their material composition, size, zeta potential, and additive content. Acute (30 min) and prolonged (up to 6 h) toxicity tests were performed to evaluate negative effects on heterotrophic and nitrifying microorganisms in activated sludge. In addition, ready and inherent biodegradability tests were performed to assess their degradation in the environment and within wastewater treatment plants, respectively. The results showed a significant acute inhibition of heterotrophic and nitrifying activity (up to 55 and 72%, respectively) at the highest concentration tested (100 mg/L, 30 min), primarily attributed to the presence of 1-dodecanol detected in the PANPs. This effect decreased with prolonged exposure, likely due to the volatilization of 1-dodecanol. Nevertheless, the PANPs were found to be non-biodegradable in both the ready and inherent biodegradability tests. Although PANPs do not appear to pose a long-term threat to the activated sludge, their persistence in the environment raises concerns about possible accumulation.