In this study, alfalfa honey and clover honey were investigated using solid-phase extraction (SPE) coupled with HPLC–MS/MS to identify and quantify plant-derived characteristic compounds. In addition, HPLC fingerprinting was established for authenticity assessment. The results showed that eight characteristic compounds were identified in alfalfa honey, mainly including terpenoids, phenolic acids, and phenolic acid esters. Among them, trans, trans-abscisic acid, cis, trans-abscisic acid, and 4-(allen-3′-yl)-3,5,5-trimethylcyclohex-2-en-1-one were identified as characteristic constituents of alfalfa honey. A total of thirteen characteristic compounds were identified in clover honey, among which kaempferol-3-O-rhamnoside and N1, N5, N10-tri-p-coumaroylspermidine exhibited strong botanical-origin specificity and could serve as potential marker compounds. The evaluation of commercial honey samples revealed that some products showed inconsistencies between their labeled floral origins and actual phytochemical characteristics. Furthermore, antibacterial activity assays demonstrated that clover honey exhibited inhibitory activity against Escherichia coli, whereas alfalfa honey showed no obvious antibacterial effect. The differences in phytochemical composition associated with floral origin may contribute to the variation in antibacterial activity between the two honey types. This study provides a theoretical basis for the botanical origin identification, quality control, authenticity assessment, and functional evaluation of alfalfa honey and clover honey.