A series of <i>N</i>-alkyl tethered C-5 functionalized bis-isatins were synthesized and evaluated for antimicrobial activity against pathogenic microorganisms. The preliminary evaluation studies revealed the compound <b>4t</b>, with an optimal combination of bromo-substituent at the C-5 position of isatin ring along with propyl chain linker being most active among the synthesized series exhibiting an IC<sub>50</sub> value of 3.72 μM against <i>Trichomonas vaginalis</i> while <b>4j</b> exhibited an IC<sub>50</sub> value of 14.8 μM against <i>Naegleria fowleri</i>, more effective than the standard drug Miltefosine. The compound <b>3f</b> with an octyl spacer length was the most potent among the series against <i>Giardia lamblia</i> with an IC<sub>50</sub> of 18.4 μM while <b>3d</b> exhibited an IC<sub>50</sub> of 23 μM against <i>Entamoeba histolytica</i>. This library was also screened against the fungal pathogen <i>Aspergillus parasiticus</i>. A number of the compounds demonstrated potency against this fungus, illustrating a possible broad-spectrum activity. Furthermore, an evaluation of these synthesized compounds against a panel of normal flora bacteria revealed them to be non-cytotoxic, demonstrating the selectivity of these compounds. This observation, in combination with previous studies that isatin is non-toxic to humans, presents a new possible scaffold for drug discovery against these important protozoal pathogens of humans and animals.