Summary How long do fungal hyphae persist in the environment? And how does this differ between groups and species of fungi? Despite growing knowledge of fungal contributions to decomposition and soil carbon cycles, surprisingly little is known about the turnover of mycelia: What happens to fungal hyphae over time? And how this impacts different fungi's contribution to carbon sequestration? In this study, we compared microscale persistence of fungal hyphae using microfluidic chip technology and visual quantification of hyphal degradation and turnover across six different wood‐decay Basidiomycete species. Measured traits included hyphal extension, coverage, turnover rates, and changes in hyphal morphology over time when supplied with two carbon sources of differing recalcitrance. Species clustered into two groups: one with a frugal nutrient strategy (high turnover capacity, active persistence of cytoplasmic hyphae) and one with a wasteful strategy (low turnover of hyphae and large remnants of skeletonized hyphae). Differences matched the ephemeral or long‐lasting nature of their fruiting bodies and the substrates they inhabit. Carbon type also influenced hyphal persistence over time. Our results suggest that hyphal turnover has a genetic basis linked to species ecology yet is also shaped by environmental factors such as carbon availability, highlighting the dynamic nature of fungal mycelia.