This study was carried out to assess the impact of pollen feeding from common floral sources in Thailand (e.g., tea, coffee, and bitter bush) on royal jelly (RJ) properties (i.e., protein pattern, (E)-9-hydroxydec-2-enoic acid (9-HDA), and (E)-10-hydroxy-2-decenoic acid (10-HDA) contents and antibacterial activity). The protein patterns from three different pollen were different, while RJ samples derived from bee colonies fed by different pollen, exhibited similar protein patterns. RJ samples from bee colonies fed by pollen from bitter bush and coffee possessed the higher 10-HDA levels than RJ collected from bee colonies fed by tea pollen. The 9-HDA was found in lower amount than 10-HDA in every sample. Even though the antibacterial activities of pollen were varied, however, RJ samples exhibited similar antibacterial properties. This is the first report showing that different pollen feeding affected 10-HDA contents, but not affected overall protein content and antibacterial properties.