This study examined the anti-inflammatory potential and metabolite profiles of Perilla frutescens var. frutescens leaf germplasm. The anti-inflammatory activity was assessed in vitro by measuring nitric oxide (NO) production and interleukin-8 (IL-8) secretion. Among the germplasm tested, PL7 and PL6 exhibited the strongest inhibitory effects on both NO and IL-8. Metabolite profiling, conducted using liquid chromatography-mass spectrometry (LC-MS), identified 60 compounds, primarily flavonoids and phenolic acids. Principal component analysis (PCA) grouped the samples into four distinct clusters: PL1–PL5, PL6, PL7, and PL8–PL10. Variable importance in projection (VIP) analysis highlighted 15 key metabolites (VIP>1.0), including tuberonic acid glucoside, caffeic acid, luteolin, and salicylic acid, which contributed to the separation of these groups. Heatmap visualization revealed distinct patterns of metabolite accumulation: vitexin and rosmarinic acid were abundant in PL1–PL5; vanillic acid-4-glucoside and prulaurasin were prominent in PL6; 5'-O-β-D-glucosylpyridoxine and esculin were concentrated in PL7; and luteolin and D-pantothenic acid were enriched in PL8–PL10. Further analysis identified key anti-inflammatory metabolites, such as apigenin-6,8-diglucoside, salicylic acid, and pinocembrin in PL6, along with quercetin-3-O-glucuronide, rosmarinic acid-3'-glucoside, and quercetin-3-O-glucoside in PL7. These findings highlight the functional diversity among perilla germplasm and their potential as valuable sources of anti-inflammatory food ingredients.