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<i>Botryosphaeriaceae</i> represents a diverse family of fungi with a cosmopolitan distribution and a wide host range. We studied species diversity and overlap of <i>Botryosphaeriaceae</i> on selected tree species of <i>Anacardiaceae</i> in disturbed (farming or forestry) and undisturbed (isolated and/or protected) ecosystems in the Limpopo and Mpumalanga provinces, South Africa. The disturbed sites resided at Tshikundamalema and Tshipise in Limpopo and the undisturbed sites at Nwanedi and the Mapungubwe National Park in Limpopo and the Kruger National Park in Mpumalanga. Asymptomatic branches were collected from <i>Mangifera indica</i>, <i>Sclerocarya birrea</i> and <i>Lannea schweinfurthii</i> trees in 2017 and 2018. Eleven species were identified using a multi-gene sequencing approach, including <i>Diplodia allocellula</i>, <i>Dothiorella brevicollis</i>, <i>Do. dulcispinae</i>, <i>Do. viticola</i>, <i>Lasiodiplodia crassispora</i>, <i>L. exigua</i>, <i>L. gonubiensis</i>, <i>L. mahajangana, Neofusicoccum parvum, Oblongocollomyces</i> sp. 1 and <i>Oblongocollomyces</i> sp. 2. Ten of the 11 species were identified in undisturbed ecosystems (eight species being unique), while only three species were identified in disturbed ecosystems (one species being unique). Two species were generalists on trees in disturbed and undisturbed ecosystems. <i>Lasiodiplodia mahajangana</i> was the most dominant species as it occurred on the three tree species of <i>Anacardiaceae.</i> Isolates of <i>N. parvum</i> occurred on both <i>S. birrea</i> (a native species) and <i>M. indica</i> (a non-native species) that occurred adjacent to each other in disturbed ecosystems, confirming the ability of this invasive pathogen to cross-infect native and non-native hosts and its abundance in human-disturbed environments. The findings from this study confirm the lack of host specificity for most species of <i>Botryosphaeriaceae</i>. The results also indicate that disturbance through human activity, such as clear-cutting, selective cutting and land-use changes, negatively influences the diversity of the <i>Botryosphaeriaceae</i>.