This study explores the potential of polymethoxyflavones (PMFs) and polyacetylated flavones (PAFs) as novel analgesic and anti-inflammatory agents. Eight derivatives, isolated from Gardenia oudiepe bud exudate or semi-synthesized from commercial kaempferol, underwent evaluations in various in vivo, in vitro, and in silico models. Acetic acid-, formalin-induced pain, and hot-plate tests were conducted in mice (n = 6). Cell viability assay, ELISA, NO measurement and protein expression by western blot were determined on RAW264.7 macrophage cells before and after exposure to LPS. Molecular docking was performed in order to putatively corroborate the affinity of the compound library to the most promising targets. Despite closely-related chemical structures, subtle modifications significantly influenced anti-nociceptive activity and affinity on diverse cellular or enzymatic targets. The library of compounds exhibited noticeable inhibitory effects on nociception in acetic acid- and formalin-induced pain assays in mice. Biochemical assays on RAW264.7 cells elucidated anti-inflammatory properties, highlighting PAFs 7 and 8 as the most active. The study indicates a peripheral anti-nociceptive profile, suggesting interferences with the production of inflammatory mediators implicated in pain disorders (e.g., COX-2, Tnf-α, IL-6 and MAPK pathway proteins). Molecular docking analyses strongly suggested interactions between PMFs/PAFs chemical library and pre-selected targets. PAFs 7 and 8 demonstrated the best binding energies, showing potential in tackling inflammation, possibly by binding to MAPK, ERK, JNK and p38. These data provide insights for lead optimization through further pharmacomodulation, paving the way for the development of innovative multi-target analgesic and anti-inflammatory drugs.