Among insects, symbionts such as bacteria and fungi can be linked to their physiology and immature development, and in some cases are part of a defense system against parasites and diseases. Current bacterial and fungal symbiont associations in solitary bees are understudied, especially in the Pacific Northwest region of the USA. We collected pollen provisions from the native spring-foraging solitary bee, Osmia lignaria Say, across two distinct foraging periods over 2 years at 22 sites along an urban-to-rural gradient in western Washington. We then used next-generation sequencing to identify bacterial and fungi within pollen provisions and assessed the effect of their richness and diversity on O. lignaria larval development success and adult emergence. We detected a significantly positive relationship between bacterial diversity in pollen with O. lignaria larval developmental success, and higher bacterial richness and diversity during the later foraging period. Fungal generic richness and diversity decreased with increasing plant richness. Although neither was associated with O. lignaria developmental success, we did detect Ascosphaera spp. known to be pathogenic to O. lignaria and other bee species. Neither bacterial or fungal richness or diversity was affected by site type when classified as urban or rural. This study provides new information on bacterial and fungal symbionts present in pollen provisions of a native solitary bee when foraging across urban and rural areas of the Pacific Northwest.