Municipal wastewater treatment plants (WWTPs) are exposed to high concentrations of micropollutants that can impact conventional activated sludge treatment. The consequences of this include failure to meet discharge standards and the disintegration of flocs, leading to poor sludge settleability. This lab-scale study focuses on the influence of doxycycline, an antibiotic widely used against human and animal diseases, on protozoa, metazoa, and bacterial communities under sludge growing conditions. Doxycycline was added to the mixed liquor of a communal WWTP up to 0, 100, 200, and 400 mg of doxycycline L−1 and incubated in batch conditions for 23 days. The regular addition of nutrient and carbon sources was preformed every 2 days to prevent sludge starvation. Sludge growth, conductivity, and settleability were measured and compared to sludge microbial community structure, determined by microscopic observations and high-throughput 16S rDNA sequencing. The high doxycycline concentration negatively impacted settleability and correlated with a decrease in bacterial diversity and floc disintegration. The addition of doxycycline promoted the enrichment of Proteobacteria Brevundimonas sp., Luteibacter anthropi, and the Bacteroidetes Chryseobacterium massoliae. These species are known to be resistant to a wide spectrum of antibiotics, including tetracyclines. A study of a larger scale may be conducted based on this study’ results.