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Sulfur is an essential plant macronutrient, and its adequate supply allows an efficient root storage and sugar extractability in sugar beets (<i>Beta</i> <i>vulgaris</i> L.). In this study, we investigated the effect of changes in sulfur availability on the endophytic community structure of sugar beets. Plants were hydroponically grown in a complete nutrient solution (S-supplied), a nutrient solution without MgSO₄ (S-deprived), and a nutrient solution without MgSO₄ for six days and resupplied with 100 μM MgSO₄ for 48 h (S-resupplied). The sulfur status was monitored by inductively coupled plasma ICP–OES, and combustion analysis together with the evaluation of microRNA395 as a biomarker for sulfate status. Metabarcoding of the bacterial 16S rRNA gene was carried out in order to determine leaf endophytic community structure. The Shannon diversity index significantly differed (<i>p</i> < 0.05) between sulfate-supplied and sulfate-deprived seedlings. Validation by Real-Time PCR showed a significant increase (<i>p</i> < 0.05) of <i>Burkholderia</i> spp. in sulfate-deprived plants as compared to sulfate-supplied ones. The study sheds new light on the effects of nutrient deficiency on the microbiome of sugar beet plants.