The kinetics and transformation pathway of acetaminophen (APAP) in natural surface water (one sample from the Yangtze River and three others from different lakes), and the changes of aquatic microbes in surface water were revealed in this study. Both photochemical and microbial reactions contributed to the transformation of APAP under irradiance of 1.0–250 mW/cm2. Microbial compositions were significantly different among surface water, and same microbial transformation product (1,4-bezoquinone) was detected as the predominant biotransformation intermediate in four studied surface water, but the lag phase (12−50-h) for the transformation was highly dependent on the aquatic microbial abundance and composition. The lag phase no longer existed with irradiance increased to 5.9 mW/cm2. Aquatic microbial abundance and composition were influenced by the presence of APAP and radiation, and the influence extent was dependent on microbial species. The findings demonstrated that the individual contribution of biotic and abiotic process to the overall transformation of APAP and maybe other phenol in surface water varied as the background composition of surface water and the external environment changed, and biotransformation dominated (>73%) the overall transformation of APAP in surface water.