There has been a growing concern on the occurrence of chiral pharmaceuticals in the aquatic environment. However, the trophic transfer of pharmaceutical enantiomers in marine organisms is still largely unknown. This study assessed the bioaccumulation and spatial distribution of four frequently detected pharmaceuticals, atenolol, metoprolol, venlafaxine, and chloramphenicol, in a subtropical marine food web in Hong Kong waters. Twenty-four species were analyzed, including mollusks, crustaceans, and fishes. Special focus was placed on the chirality of the four analytes comprising ten different stereoisomers. Results showed that mean concentrations of individual pharmaceuticals ranged from <0.03 to 5.88 ng/g wet weight, and invertebrates generally had higher pharmaceutical concentrations than fishes. Organisms from Hong Kong western waters were likely more contaminated by the studied pharmaceuticals than those from southern and eastern waters. Trophic dilution was observed for atenolol and chloramphenicol, with the trophic magnification factors of 0.164 and 0.517, respectively. R-(+)-atenolol, S-(−)-metoprolol, and R-(−)-venlafaxine were enantioselectively accumulated in fishes, and stereoisomeric impurities of chloramphenicol, i.e., enantiomers apart from R,R-para-form, were widespread in the investigated species. Under the worst-case scenario, atenolol and metoprolol in sampled fishes might exceed toxic threshold, while local adults were unlikely to experience health risks from the exposure to the studied pharmaceuticals via seafood consumption.