Winemakers have long used copper as a conventional fungicide treatment on grapevine and only recently, the use of biocontrol agents depicted a promising alternative. Most of the studies that investigate the impact of fungicide treatments, focus on specific pathogenic microbes. In the present work instead, a field experiment conducted in South Africa shows the seasonal microbial change occurring on grapevine leaves, periodically treated with two different fungicide treatments: copper sulphate and Lactobacillus plantarum MW-1. In this work, NGS data were combined with strain-specific and community qPCRs to reveal the shift of the microbial communities throughout the growing season and highlight the impact of fungicides on the microbiota. Only the family of Lactobacillaceae systematically changed between treatments, while the bacterial community remained relatively stable over time. MW-1 was exclusively detected on biocontrol-sprayed leaves. Conversely, the fungal community was largely shaped by sampling time, underlining the succession of different dominant taxa over the months. Between treatments, only a few fungal taxa appeared to change significantly and the fungal load was also comparable. Monitoring the dynamics of the microbial community under different fungicide treatments may advise the best timing to apply treatments to the plants, toward the realization of more sustainable agricultural practices.