The heat shock protein 90 (Hsp90) family plays a critical role in maintaining the homeostasis of the intracellular environment for human and prokaryotic cells. Hsp90 orthologues were identified as important target proteins for cancer and plant disease therapies. It was shown that gambogic acid (GBA) has the potential to inhibit human Hsp90. However, it is unknown whether it is also able to act on the bacterial high-temperature protein (HtpG) analogue. In this work, we screened GBA and nine other novel potential Hsp90 inhibitors using a miniaturized high-throughput protein microarray-based assay and found that GBA shows an inhibitory effect on different Hsp90s after dissimilarity analysis of the protein sequence alignment. The dissociation constant of GBA and HtpG Xanthomonas (XcHtpG) computed from microscale thermophoresis is 682.2 ± 408 μM in the presence of ATP, which is indispensable for the binding of GBA to XcHtpG. Our results demonstrate that GBA is a promising Hsp90/HtpG inhibitor. The work further demonstrates that our assay concept has great potential for finding new potent Hsp/HtpG inhibitors.