New synthetic opioids (NSOs) are one of the fastest growing groups of new psychoactive substances. Amid this dynamic landscape, insight into the pharmacology of NSOs is important to estimate the harm potential of newly emerging drugs. In this work, we determined the mu-opioid receptor (MOR) affinity and activation potential of seven poorly characterized non-fentanyl NSOs (N-ethyl-U-47700, 3,4-difluoro-U-47700, U-47931E/bromadoline, 2,4-difluoro-U-48800, U-62066/spiradoline, 2F-viminol, ketobemidone) and a panel of nine reference opioids. MOR affinity was determined via [H-3]-DAMGO binding in rat brain tissue homogenates, and was found to correlate well with different functional parameters. MOR activation potential was studied at different levels of receptor signaling using three distinct assays (NanoBiT (R) MOR-beta-arrestin2/mini-G(alpha i) and AequoScreen (R)). The most active compounds were ketobemidone (EC50 32.8-528 nM; E-max 105-271%, relative to hydromorphone) and N-ethyl-U-47700 (EC50 241-767 nM; E-max 139-247%). The same opioids showed the strongest MOR affinity. As most of the other NSOs only weakly activated MOR in the three assays (EC50 values in the high nM-mu M range), they likely do not pose a high overdose risk. 2F-viminol (EC50 2.2-4.5 mu M; E-max 21.2-61.5%) and U-47931E/bromadoline (EC50 0.55-2.9 mu M; E-max 52.8-85.9%) were partial agonists compared to hydromorphone, and maximum receptor activation was not reached for 2,4-difluoro-U-48800 (EC50 > 22 µM). We further highlight the importance of considering specific assay characteristics upon interpretation of potencies, efficacies and biased agonism. As absolute values may greatly differ between assays with varying experimental set-ups, a comparison of functional parameters to those of well-characterized reference agonists is considered the most informative.