To effectively minimize biofilms, which are prevalent in chlorinated drinking water distribution systems, the effect of biofilm age on antimicrobial tolerance and resistance must be investigated. It was our hypothesis that antimicrobial tolerance emerges quickly during biofilm development and that both antimicrobial tolerance and resistance increase as the biofilm ages. We further hypothesized that antimicrobial tolerance and resistance vary among the individual community members. In this study, young and mature biofilms of Pseudomonas aeruginosa, a model biofilm organism, were grown and exposed to antimicrobial agents in several different treatments. Results showed that the increased antimicrobial tolerance of intact biofilms compared to planktonic cells arises early (i.e., within hours) in biofilm development. The short-term tolerance of resuspended mature biofilm cells to antimicrobial agents peaked at a biofilm age of 14 days and subsequently declined; the peak and decline in antimicrobial tolerance may be related to periodic detachment events in the biofilm. The antimicrobial resistance of resuspended mature biofilm cells continuously exposed to antimicrobial agents increased with biofilm age. Furthermore, individual members in mature biofilm communities exhibited variation in antimicrobial tolerance, thereby highlighting divergence of the biofilm community from the original parent strain.