ObjectivesExamine the association between multiple clinical staff levels and case-mix adjusted patient mortality in English hospitals. Most studies investigating the association between hospital staffing levels and mortality have focused on single professional groups, in particular nursing. However, single staff group studies might overestimate effects or neglect important contributions to patient safety from other staff groups.DesignRetrospective observational study of routinely available data.Setting and participants138 National Health Service hospital trusts that provided general acute adult services in England between 2015 and 2019.Outcome measureStandardised mortality rates were derived from the Summary Hospital level Mortality Indicator data set, with observed deaths as outcome in our models and expected deaths as offset. Staffing levels were calculated as the ratio of occupied beds per staff group. We developed negative binomial random-effects models with trust as random effects.ResultsHospitals with lower levels of medical and allied healthcare professional (AHP) staff (e.g, occupational therapy, physiotherapy, radiography, speech and language therapy) had significantly higher mortality rates (rate ratio: 1.04, 95% CI 1.02 to 1.06, and 1.04, 95% CI 1.02 to 1.06, respectively), while those with lower support staff had lower mortality rates (0.85, 95% CI 0.79 to 0.91 for nurse support, and 1.00, 95% CI 0.99 to 1.00 for AHP support). Estimates of the association between staffing levels and mortality were stronger between-hospitals than within-hospitals, which were not statistically significant in a within–between random effects model.ConclusionsIn additional to medicine and nursing, AHP staffing levels may influence hospital mortality rates. Considering multiple staff groups simultaneously when examining the association between hospital mortality and clinical staffing levels is crucial.Trial registration numberNCT04374812.