The 2016 Winston-Salem Open was a men's tennis tournament played on outdoor hard courts. It was the 48th edition of the Winston-Salem Open (as successor to previous tournaments in New Haven and Long Island), and part of the ATP World Tour 250 Series of the 2016 ATP World Tour. It took place at Wake Forest University in Winston-Salem, North Carolina, United States, from August 23 through August 29, 2015. It was the last event on the 2016 US Open Series before the 2016 US Open.
| Country | Player | Rank* | Seed | |
|---|---|---|---|---|
 France | 15 | 1 | ||
 Spain | Roberto Bautista Agut | 17 | 2 | |
| 19 | 3 | |||
| 23 | 4 | |||
| 24 | 5 | |||
| 29 | 6 | |||
| France | Gilles Simon | 31 | 7 | |
| Spain | 33 | 8 | ||
 Serbia | 35 | 9 | ||
| 36 | 10 | |||
 Italy | 40 | 11 | ||
 Russian Federation | 41 | 12 | ||
 Argentina | 43 | 13 | ||
 Cyprus | 44 | 14 | ||
| Spain | 45 | 15 | ||
| Spain | 48 | 16 |
The following players received wildcards into the singles main draw:
The following player received entry using a protected ranking:
The following players received entry from the qualifying draw:
The following player received entry as a lucky loser:
Daniel Evans Marcel Granollers →replaced by 
Víctor Estrella Burgos Pierre-Hugues Herbert Nicolas Mahut →replaced by 
Lukáš Rosol Juan Mónaco →replaced by Evgeny Donskoy Benoît Paire →replaced by 
Damir Džumhur Guido Pella →replaced by Stéphane Robert
Dudi Sela →replaced by Jan-Lennard Struff Horacio Zeballos →replaced by Tim van Rijthoven| Country | Player | Country | Player | Rank1 | Seed | |
|---|---|---|---|---|---|---|
 Poland | Łukasz Kubot | Serbia | Nenad Zimonjić | 58 | 1 | |
| Mate Pavić |  New Zealand | Michael Venus | 78 | 2 | ||
 Sweden | Robert Lindstedt |  Pakistan | Aisam-ul-Haq Qureshi | 79 | 3 | |
| Eric Butorac | Scott Lipsky | 86 | 4 |
The following pairs received wildcards into the doubles main draw:
See main article: 2016 Winston-Salem Open – Singles.
Pablo Carreño Busta def. Roberto Bautista Agut, 6–7(6–8), 7–6(7–1), 6–4See main article: 2016 Winston-Salem Open – Doubles.
Guillermo García López / 
Henri Kontinen def. Andre Begemann / 
Leander Paes, 4–6, 7–6(8–6), [10–8]
