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This study evaluated activities of crude extracts from different parts of the tree of heaven (Ailanthus altissima (Mill.) Swingle) collected in Slovenia and Hungary, using effect-directed analyses based on hyphenation of high-performance thin-layer chromatography (HPTLC) and nine in vitro assays performed in situ on chromatographic plates after the separation. HPTLC separation combined with a set of four antibacterial assays, two antifungal assays, and three enzyme inhibitor assays to evaluate the extracts of 14 plant parts: young shoots, young leaves, mature leaves, yellow leaves, petioles of leaves, petioles of male inflorescences, petioles of fruits, female inflorescences, male inflorescences, mature male inflorescences, bark of 1–2-year branches, bark of 2-year branches, bark of tree trunk, and bark of roots. Antibacterial activities against Gram-positive bacteria (Bacillus subtilis, Rhodococcus fascians) and Gram-negative bacteria (Aliivibrio fischeri, Pseudomonas syringae pv. maculicola (Psm)), as well as inhibition of enzymes α-glucosidase, lipase, and acetylcholinesterase, were observed for all extracts. Extracts differed in their antifungal activities. Extracts of young shoots, mature leaves, petioles of leaves, and bark of roots showed antifungal activity against plant pathogens Fusarium avenaceum and Bipolaris sorokiniana. Extracts of yellow leaves, male inflorescences, bark of 1–2-year branches, and bark of tree trunks were only active against F. avenaceum, whereas extracts of young leaves were only active against B. sorokiniana. This study is the first to report that A. altissima extracts exhibit (1) antifungal activity against F. avenaceum and B. sorokiniana; (2) antibacterial activity against A. fischeri, Psm, R. fascians, and B. subtilis (except leaves, bark of branches and bark of tree trunks); and (3) inhibitory activity toward lipase, α-glucosidase (except bark of tree trunks), and acetylcholinesterase (except bark of tree trunks).