Codeine, an opium alkaloid, was transformed in aerobic batch experiments with activated sludge into several transformation products (TPs). For eight TPs, the chemical structures were unambiguously identified by a multistep approach using results from high-resolution mass spectrometry (HR-MS) and 1D and 2D nuclear magnetic resonance (NMR) experiments. For an additional 10 TPs, tentative structures were proposed. Most of the TPs identified exhibited only slightly modified molecular structures featuring double bond shifts, introduction of hydroxy groups, or amine demethylation. The transformation pathway of codeine in contact with activated sludge is characterized by a combination of biologically and chemically mediated reactions. Biological oxidation of codeine leads to the formation of the α,β-unsaturated ketone codeinone, which is the precursor for further abiotic and biotic transformation due to its high chemical reactivity. An analytical method based on solid-phase extraction and LC tandem MS detection was developed to confirm the formation of several TPs in wastewater treatment plants (WWTPs). The mass balances were comparable to those obtained from batch experiments. An HR-MS screening approach of TPs from dihydrocodeine and morphine revealed that the knowledge from the codeine transformation pathway can be extrapolated to the distinct transformation pathways of these structurally related opium alkaloids. In total, 17 TPs were proposed for morphine and 2 TPs for dihydrocodeine.