English and Welsh red squirrel (Sciurus vulgaris) populations are often small, geographically isolated and particularly vulnerable to stochastic events, such as extirpation due to infectious disease. Immigration or emigration via connected habitat with later re-colonisation may be restricted or not possible and pathogenic disease impact magnified, increasing population loss risk compared to extensive habitat populations. In the United Kingdom (UK), both squirrelpox virus (SQPV) and adenovirus (ADV) infections are major threats to regional in situ conservation and translocation studies. It is important that any amplification of mortality impacts are understood including via population size, isolation and additional threats such as urbanisation. Here, we quantified mortalities between August 2019 and July 2020 in three small isolated English woodland populations and a small woodland fragment on the island of Anglesey, Wales. A cumulative red squirrel population estimate at the four sites during the study was 45–55 animals, with 30 recorded deaths and 15 carcasses recovered for post-mortem examination. Cause of death varied, including trauma, predation, ectoparasite burden, ADV-associated disease and squirrelpox. Negative-stain transmission electron microscopy (TEM) confirmed squirrelpox in four animals, with ADV-associated disease recorded in five others. Polymerase chain reaction (PCR) confirmed asymptomatic ADV infection in three further animals. Surveillance throughout the study revealed red squirrel persistence in each woodland location with grey squirrels (Sciurus carolinensis) recorded at two of four sites. Proactive management action, including red squirrel conservation mitigations have been prepared for each site, with formal red squirrel strategy development for such isolated populations ongoing, via citizen-based action and appropriate analytical support. We recommend analytical disease surveillance and invasive species management continue as central pillars of such conservation efforts.