We report new helium and carbon isotope (3He/4He and δ13C) and relative abundance (CO2/3He) characteristics of a suite of 20 gases and fluids (cold mazuku-like CO2 vents, bubbling mud-pots, hot and cold springs) from 11 different localities in Rungwe Volcanic Province (RVP), southern Tanzania and from 3 additional localities in northern Tanzania (Oldoinyo Lengai Volcano and Lake Natron). At RVP, fluids and gases are characterized by a large range in He-isotope compositions (3He/4He) from 0.97 RA to 7.18 RA (where RA = air 3He/4He), a narrow range in δ13C ratios from − 2.8 to − 6.5‰ (versus VPDB), and a large range in CO2/3He values spanning nearly four orders of magnitude (4 × 109 to 3.2 × 1013). Oldoinyo Lengai possesses upper‐mantle-like He–CO2 characteristics, as reported previously (Fischer et al., 2009), whereas hot springs at Lake Natron have low 3He/4He (~ 0.6 RA), CO2/3He (~ 5–15 × 108) and intermediate δ13C (~−3.7 to − 4.9 ‰). At RVP, fluid phase samples have been modified by the complicating effects of hydrothermal phase-separation, producing CO2/3He and δ13C values higher than postulated starting compositions. In contrast, gas-phase samples have not been similarly affected and thus retain more mantle-like CO2/3He and δ13C values. However, the addition of crustal volatiles, particularly radiogenic helium from 4He-rich reservoir rocks, has modified 3He/4He values at all but the three cold CO2 gas vent (i.e., mazuku) localities (Ikama Village, Kibila Cold Vent and Kiejo Cold Vent) which retain pristine upper-mantle He-isotope (~ 7 RA) and He–CO2 characteristics. The extent of crustal contamination is controlled by the degree of interaction within the hydrothermal system, which increases with distance from each major volcanic center. In contrast, we propose that pristine cold CO2 mazuku gases collected at stratigraphic contacts on the flanks of RVP volcanoes may potentially tap isolated gas pockets, which formed during previous eruptive events and have remained decoupled from the local hydrothermal system. Furthermore, by identifying and utilizing unmodified gas samples, we determine mantle versus crustal provenance of the CO2, which we use to estimate mantle-derived CO2 fluxes at both Rungwe and Lake Natron. Finally, we investigate the origin of the apparent discrepancy in He isotopes between fluids/gases and mafic phenocrysts at RVP (from Hilton et al., 2011), and discuss the tectonic (i.e., rift zone dynamics) and petrogenic conditions that distinguish RVP from other plume-related subaerial rift zones.