Microthrix parvicella is a filamentous bacterium that frequently causes severe bulking events in wastewater treatment plants (WWTPs) worldwide. In this study, sludge properties and dynamics of filamentous bacteria in a Beijing WWTP seasonally suffering from M. parvicella bulking were continuously monitored over a duration of 15 months, and the correlations between M. parvicella and operating parameters were evaluated. The predominance of M. parvicella was observed at low temperatures (14–18.8 °C) with the relative abundance of around 30% (estimated by both qPCR and FISH analysis). Using micromanipulation technology, 545 filaments of M. parvicella were micromanipulated from bulking sludge (SVI > 180 mL g−1) on six different media. After 3-month purification and enrichment, six strains, phylogenetically closely related to Candidatus Microthrix parvicella, were successfully acquired on R2A medium (20 °C) in pure cultures. Considering the limitation and extremely slow growth rate of M. parvicella filaments, newly isolated strains represent valuable sources for further investigations on the physiology and behavior of this filamentous bacterium, with the focus on the establishment of bulking control strategy.